This experiment can be used to investigate the following
concepts and phenomena:

1. To learn how to grow yeast, to change their environment, and to observe them through the microscope.

2. To observe and learn to identify the characteristic shapes (morphology) of yeast cells at important stages of their sexual
life cycle.

3. To follow the inheritance of a color trait (pink colony vs. cream colored colony) and see if one variation is dominant and
the other recessive, or whether intermediate colors are
inherited.

The yeast life cycle may be used to teach a variety of
concepts. (See the Cycles in the Life of Yeast and A Simple
Cross and Cell Division Cycle segments in video tape I.) As the
students gain skills in manipulating the yeast you may wish to
have them go through the cycle several times using different
haploid strains. This particular protocol uses HA2 (pink) and
HBT (cream) haploid strains. Microscopes are used to observe the
characteristic shapes of the stages of the yeast life cycle.
Color changes are used to develop an understanding of dominant
and recessive phenotypes.

You may wish to show a variation on genetic behaviour by
crossing various strains. For example, HA2 (pink) and HB1 (pink)
produce cream colored diploids when the two haploids are mated.
It is possible to use these strains to demonstrate that when
gametes combine, the offspring sometimes look different than
either parent.

Whan HA2 & HB1 mate, they mark the transition between haploid
and diploid with a color change (haploids pink; diploids cream).
This feature may be useful if microscopes are not available to
examine the cell shape change that also marks the transition from
haploid to diploid.

Getting Ready:

You may wish to prepare this first set of plates for the
students. Yeast strains usually come from the supplier growing
on agar slants. Contamination may be a problem when students use
the master set of yeast strain slants as the source of their
strains. A quick method for preparing the "Getting Ready" plates
is for the teacher to make a master plate, incubate it overnight,
and then use the replica plating method to make copies for the
students. (See Replica Plating segment in video tape III.)
Student Data Record Sheet
If you want your students to do the "Getting Ready" step of the
experiment, you can subculture the strains on to YED plates. One
subculture plate of each mating type will supply enough yeast for
all the students. If several groups need access to the yeast at
the same time, you may want to make several subculture plates
(See video tape segment Subculturing Yeast.)

You may wish to show your students the video tape segment
Subculturing Yeast which demonstrates the use of sterile
toothpicks for moving yeast cells.
If you collect the used toothpicks in small beakers, the
toothpicks can be rinsed, brushed, and resterilized in an
autoclave or pressure cooker and used again.

Mating Two Haploid Strains and Observing Zygotes:

4. If your students keep a lab journal you may wish to have them
record data and drawings as journal entries. An alternative
method is to copy and hand out the Data Record Sheet provided
with these materials.

5. In order to view the cell shapes, the students need to use
the high power lens of their microscopes; 400X is adequate.
If streaming of the cells in the water currents under the
coverslip is a problem you may wish to seal the coverslip
with fingernail polish. (See video tape segment Wet Mount
Slides.) You can display the slides on a video monitor using
a video microscope. It is possible to use a home video
camera and TV monitor for this purpose. (See Microscope and
Home Video Camera segment in video tape III.)

You can use a microscope to observe cells directly on the agar
surface by gently dropping a cover slip onto the area to be
observed. Focus the microscope through the cover slip just as
you would on the slide. Naturally, the plate is almost certain
to become contaminated!

Time Shifting:

Rather than refrigerating all the plates after 3 hours, you
may wish to prepare a refrigerated mating mixture in advance for
the students to use to make their mating mixture wet-mount
slides.

If you have a "premated" mixture available, the students
should be able to complete "Mating Two Haploid Strains and
Observing Zygotes" in one 50 minute lab period. If your students
are expected to observe their own mating mixtures, you will need
to refrigerate their plates so they will be able to observe the
characteristic mating shapes the next day. You will also need to
adjust the time line for your class.

The time line assumes that you have prepared a premated
mixture for the students to observe on the same day that they
make their own mating mixture.
The point to remember as you work out the logistics for your
class is that it takes about three hours at 30o C for mating to
occur and for zygotes to become visible. Refrigeration will
delay the process.

Selecting the Diploids:

In this experiment the students use complementation to select
diploid cells. HA2 carries the ade2 mutation but it carries a
functional
TRP5 gene. It is not able to synthesize adenine due to the ade2
mutation but it is able to synthesize tryptophan. The absence of
adenine in the growth medium prevents HA2's growth on MV. HBT
carries a functional ADE2 gene but it carries the trp5 mutation.
It is not able to synthesize tryptophan but it is able to
synthesize adenine. The absence of tryptophan in the growth
medium prevents HBT's growth on MV. When the two haploid cells
mate and fuse, the resulting diploid cell has one functional copy
of the ADE2 gene and one functional copy of the TRP5 gene. It is
able to synthesize both adenine and tryptophan thus allowing the
diploid to grow on MV medium.

Genotype of haploid parents

mutant

functional

HA2

ade2

TRP5

HBT

trp5

ADE2

Genotype of diploid cells
Each diploid cell has one functional copy of each gene.

HA2/HBT TRP5/trp5 ade2/ADE2

Selecting the diploids:

You may wish to use the replica plating technique for this
step. (See Replica plating segment in video tape III)

Presporulation:

1. The diploid cells growing on MV should be cream-colored.

Sporulating the Diploids:

1. The diploid cells growing on YED should remain cream-colored.

2. Since the diploid cells don't divide rapidly on YEKAC, be
sure to transfer enough cells so that you have plenty to
produce asci. For some diploid strains, sporulation
efficiency is affected by cell density. If you don't find
asci in one section of the streak, check sections of the
streak with higher and lower cell densities.

Observation of Asci and Germination of Spores:

2. Some strains take longer than three days to sporulate. The
diploid cells produced in the HA2 x HBT cross tend to
sporulate quickly. For example, if you put the diploid cells
on YEKAC on Friday you should have some spores by Monday.

4. This technique is routinely used to spread cells out far
enough so that a single cell can produce a single isolated
colony. The ascus wall is tough and tends to hold the spores
together. In many cases even after spreading the cells, there
will still be more than one spore stuck together. As noted
in the experimental procedure, some of the spores will be
mating type a and some mating type .
After they germinate,
they may mate and produce diploid colonies. If both the
spores happen to carry the ade2 mutation, the resulting
diploid will be homozygous for ade2 and have a pink
phenotype. In other cases, the spores may be too far apart
from one another to mate. These spores will form haploid
colonies. If the spore carries the ADE2 allele of the gene,
the colony will be cream-colored. If the spore carries the
ade2 allele of the gene, the colony will be pink. You may
wish to pick samples of the pink colonies, subculture them
overnight on YED and then transfer them to YEKAC as a test of
ploidy. Diploid cells will sporulate and haploid cells will
not.

Looking for the Missing Color:

The reappearance of the pink phenotype demonstrates that the
complete sexual life cycle has been completed.

Objectives and Applications:

Our understanding of the nature of genes at the molecular
level came from the concept of allelism, that mutations can occur
at many places in a gene and there are multiple genes that
contribute to the same phenotypic trait. Mutation to a red
colony color have been found in two different genes in the
biosynthetic pathway for adenosine monophosphate (AMP), ade1 and
ade2. When students can understand these relationships, the can
appreciate the concept of allelism and how we learned what genes
are. (See The Two Gene Hypothesis and A Genetic Test For Allelism
segments in video tape I).

Objectives:
1. To make the four possible crosses between two different red
mutants and determine the color and growth requirements of
each of the diploids.
2. To determine whether the results support the hypothesis that
the two red mutants have mutations in different genes.
3. To see that this experiment provides a test for whether two
mutations affect the same or different genes (a test for
allelism).

Subculture Parent Strains:
You may wish to prepare this first set of plates for the
students. Yeast strains usually come from the supplier growing
on agar slants. Contamination may be a problem when students use
the master set of yeast strain slants as the source of their
strains. A quick method for preparing these subculture plates is
for the teacher to make a master plate, incubate it overnight,
and then use the replica plating method to make copies for the
students. (see Replica Plating segment in video tape III.)
These student copies need to be incubated overnight before the
strains are mated.

If you want the students to do this step of the experiment,
you may wish to subculture the strains on YED plates. One
subculture plate of each strain will supply enough yeast for all
the students. If several groups need access to the yeast at the
same time, you may want to make several subculture plates of each
strain. (See Subculturing Yeast segment in video tape III.)

Numbers refer to steps in the student procedure.
Cross all four strains:

Remind the students to use a new sterile
toothpick for each
different strain and to keep the spots from touching each
other. If individual students don't get the expected results
from the crosses keep in mind the possibility that they may
have cross contaminated the strains during this step.

If the haploid cells don't get thoroughly mixed
there will be
areas of unmated cells on the edge of the mating mixture.
These unmated areas will show the phenotype of the haploid
parent rather than the phenotype of the diploid cells. For
thorough mixing the mating mixture should cover an area
larger than the two spots of haploid
yeast.

The students are asked to record only color and growth on MV.
They will not be confirming the mating phenotype of the diploid
cells. It is possible to take the diploid cells produced in this
exercise through the life cycle if you want to follow the
segregation patterns of mating types, ADE1 and ADE2.

The expected data supports the two gene hypothesis. A
functional copy of both genes is necessary to produce the normal
cream colored colonies. The genes code for different enzymes in
the same biochemical pathway (AMP synthesis).

Complementation, allelism, and defining a gene:

Genetics is the study of genes. Genes carry the information
that defines every organism. To accomplish this, genes (with the
help of the cells they live in) must do three things:

1. They must reproduce: their information must be copied
faithfully.
2. They must be transmitted: their information must be passed
precisely to new cells.

3. They must act: their information must result in metabolic
reactions and cellular structures.

Let's think about a life cycle experiment where you cross two
red haploid strains together and get a diploid that is cream
colored. When you sporulate the diploid you recover both red and
cream colonies. A model (theory) developed by Gregor Mendel to
explain inheritance of different traits in peas might explain
these results. It is called the recessive-dominant theory.
Suppose that in every normal yeast cell there are two genes--for
now call them GENE1 and GENE2--that are needed for the colonies
to be cream colored. If one of the red strains had a mutant form
of one of the genes and the other strain had a mutant form of the
other gene, we could explain the results of the cross and make
some predictions that you could test in another experiment.
Let's assume that the mutant genes are damaged forms of the
normal ones and for now call the mutant forms gene1 and gene2.
We could say that one of the red parent strains contained GENE1
and gene2 and the other parent gene1 and GENE2. The diploid
formed between them could then be represented as follows:

GENE1gene2gene1GENE2

From this you can see that there is one copy of each of the
normal genes (capital letters) and one copy of each of the mutant
genes (lower case letters). If the normal genes still work in
the presence of the mutant genes, they can still do whatever it
is that makes the colony cream colored. The mutant genes are
just going along for the ride.

Let's see if there is some way we can test this model. What
would happen if you crossed two red haploid strains that had the
same mutant gene, such as crossing a gene1 GENE2 with another
gene1 GENE2. Then the diploid would be:

gene1GENE2gene1 GENE2

This diploid has no copies of the normal GENE1, so we would
expect it to be red. In the same way, if you crossed GENE1 gene2
by another GENE1 gene2 strain you would get

GENE1gene2GENE1 gene2

which has no copies of the normal GENE2, so it should also be
red.
To summarize this, we can say that when mutations affect
different genes, whethere the same phenotype or not, they will
complement each other; when crossed together the diploid will
have the normal phenotype. This demonstrates that they are not
alleles. When they are in the same gene, they will fail to
complement each other and that is taken as evidence that they are
alleles. So complementation provides a genetic test for allelism
and gives a criterion for determining whether two mutations
affect the same or different genes, and therefor, different
functions. Complementation also provides a tool for selecting
diploids from a mating mixture. This tool was used in A Simple
Cross.

Comments on genetic nomenclature:

The following tables show the shorthand strain numbers, the
genotypes in the official Yeast Genetics nomenclature, and a
description of the phenotypes for the strains used in this
experiment. This experiment, which shows that mutations in two
different genes can give similar phenotypes, illustrates why this
more elaborate nomenclature is necessary.

Objectives and Applications:

This experiment is designed to be used in conjunction with
the traditional textbook treatment of a simple dominant/recessive
two-factor cross. It gives graphical realism to the ever-popular
Punnett's Square. (See A Traditional Dihyrid Cross segment in
video tape I).

Objectives:
1. To see first-hand how the genotypes of the haploid gametes
combine to form the phenotypes of the diploid offspring for
dominant and recessive alleles.

2. To see that there is no uncertainty in the outcome
(phenotypes of the offspring) when there is no uncertainty in
the genotypes of the gametes. In the diploid dihybrid cross
the uncertainty is the result of random sampling of the pool
of gametes from the parental cross.

Yeast strains usually come from the supplier growing on agar
slants. Contamination may be a problem when students use the
master set of yeast strain slants as the source of their strains.
You may wish to subculture the strains on YED plates. One
subculture plate containing all eight haploid strains will supply
enough yeast for a class. If several groups need access to the
yeast at the same time, you may want to make several subculture
plates (See Subculturing Yeast segment in video tape III).
Alternatively you may wish to prepare the Day 1 plates for the
students. A quick method for preparing these plates is for the
teacher to make a master plate, incubate it overnight, and then
use the replica plating method to make copies for the students
(see Replica Plating segment in video tape III).

See the Laboratory Methods section for agar media recipes.
You will need to make the plates several days in advance.

Each student or team will require at least 50 sterile
toothpicks for this experiment. Toothpicks are sterile from the
box and there are 750 toothpicks in each box. (See Laboratory
Methods F: Using Toothpicks and Inoculating Loops)

Each student or team will require at least 1 mating grid. A
copy master of six grids is included with these materials.

1st Day:
Have the students write the expected genotypes and phenotypes
on the mating grid and then tape the grid to the bottom of the
YED plate as a pattern for plating the yeast.
Only the eight haploid strains are plated on Day 1.

2nd Day:
Stress that fresh toothpicks should be used for each
different strain or mating mixture. The best results are
obtained when only small amounts of yeast are used to make each
mating mixture.

3rd Day:
Once again the best results are obtained when only small
amounts of yeast are transferred during replica plating. If you
use replica plating equipment you will need a fresh sterile
velvet for each plate that is copied.

4th Day:
If the mixtures were not mixed thoroughly there may be pink
areas in otherwise cream-colored mixtures. These mixtures should
be scored as cream-colored.

Comments on genetic notation:
We have chosen to use traditional notation for the genotypes
in this experiment so that it will be more consistent with usual
textbook treatments of the dihybrid cross and Punnett's Square.
In later experiments we will introduce the more sophisticated
notation of modern yeast genetics. Even so, you have some
options for how much you want to simplify things.

Case 1

We can represent the hypothetical dihybrid cross between two
diploids as follows:

RRtt x rrTT

But in yeast, we don't actually cross diploids. We first
sporulate these diploids, obtaining two types of spores, each in
both mating types:

ARt, ArT and BRt, BrT

These represent the gametes in the parental cross. We then
mate either ARt x BrT or BRt x ArT to obtain the F1 diploids,
which would be:

F1: ABRrTt

If we sporulate this diploid, we get the following eight
haploid genotypes (spores or gametes) shown on the previous page.

In this experiment the students start here to obtain the F2
generation, without the complication of statistical sampling, by
setting up the following grid of crosses:

F2:

ART
ARt
ArT
Art

BRT

c+
c+
c+
c+

BRt

c+
c-
c+
c-

BrT

c+
c+
r+
r+

Brt

c+
c-
r+
r-

where c = cream, r = red, + = grows on MVA, and - = doesn't
grow on MVA
Case 2
In yeast, mating type is controlled by the mating type genes
that segregate as alternative Mendelian alleles, so it seems
natural to include them in the formal description of the
genotypes, as we have done above. However, it may simplify the
discussion to treat mating type separately. The A's and B's
could, therefore, be omitted in all the above genotypes. If, for
example, you describe a cross as Rt x rT, it is implicit that
they are different mating types. If this approach is taken, then
the whole scheme simplifies to the following:

P1: RRtt x rrTT

gametes: Rt and rT

F1/P2: RrTt

F2:

RT
Rt
rT
rt

RT

c+
c+
c+
c+

Rt

c+
c-
c+
c-

rT

c+
c+
r+
r+

rt

c+
c-
r+
r-

Comments:
The main difference between this experiment and the analogous
case in higher organisms, is that in yeast we have control of the
life cycle so that we can deal directly with the haploid stages.
We can isolate, as pure strains, the haploid parents of the F2
generation, which correspond to the gametes from the P2 parents.
By being able to identify these, isolate them as pure strains,
and make all the possible crosses, we remove all the chance
factors. In higher organisms, we hypothesize the segregation of
alleles in a hybrid when it goes through meiosis. If we make two
assumptions, 1) that the alternative alleles segregate
independently into the gametes, and 2) that there is
dominant/recessive expression of the respective alleles, then we
can predict the 9:3:3:1 ratios among the F2 offspring.
Accordingly, this experiment provides a direct demonstration of
how the diploid phenotypes are derived from the genotypes of the
parents.

This does not demonstrate independent segregation, which
requires showing that the haploid phenotypes did, in fact, occur
in equal numbers among the spores from the F1 diploids.

In the context of animal breeding, or even human genetics,
what we do with yeast when we isolate two haploids and mate them
has a parallel in in vitro fertilization. However, we do not yet
have the ability to score many genetic traits in gametes of
higher forms, nor can we propagate them asexually.

Genetic test for Allelism

Objectives and Applications:

This is a variation on Traditional Dihybrid Cross and Two
Genes/One Trait experiments. It can serve as a briefer
substitute for both or it can be used as a supplement. In the
former case you may want to introduce some of the ideas developed
in those experiments. (See The Two Gene Hypothesis and A Genetic
Test For Allelism segments in video tape I).

Objectives:
1. To have the students see how the pattern of phenotypes is
altered in a dihybrid cross when both mutations yeild the
same phenotype compared with the traditional case of two
different phenotypes.

2. To see how complementation provides a way to distinguish
between non allelic mutants that have the same phenotype.

Yeast strains usually come from the supplier growing on agar
slants. Contamination may be a problem when students use the
master set of yeast strain slants as the source of their strains.
You may wish to subculture the strains on YED plates. One
subculture plate containing all eight haploid strains will supply
enough yeast for a class. If several groups need access to the
yeast at the same time, you may want to make several subculture
plates (see Subculturing Yeast segment in video tape III).
Also, you may wish to prepare the 1st Day plates for the
students. For a quicker preparation of these plates, the
teacher can make a master plate, incubate it overnight, and then
use the replica plating method to make copies for the students
(see Replica Plating segment in video tape III).

See the Laboratory Methods section for agar media recipes.
You will need to make the plates several days in advance.

Each student or team will require at least 50 sterile
toothpicks for this experiment. Toothpicks are sterile from the
box.

Each student or team will require at least 1 mating grid. A
copy master of six grids is included with these materials.

Day 1:
Have the students write the expected genotypes and phenotypes
on the mating grid and then tape the grid to the bottom of the
YED plate as a pattern for plating the yeast.

Only the eight haploid strains are plated on Day 1.

Day 2:
Stress that fresh toothpicks should be used for each
different strain or mating mixture. The best results are
obtained when only small amounts of yeast are used to make each
mating mixture.

LDay 3:
Once again the best results are obtained when only small
amounts of yeast are transferred during replica plating. If you
use replica plating equipment, you will need a fresh sterile
velvet for each plate that is copied.

Day 4:
If the mixtures are not mixed thoroughly there may be pink
areas in otherwise cream-colored mixtures. These mixtures should
be scored as cream-colored.

The pattern that results should represent the Punnett Square
as it is used to diagram the dihybrid cross. The pattern of
dominance of ADE1 and ADE2 over ade1 and ade2, respectively,
should also be clear. This case is slightly different from the
usual textbook dihybrid , since the two mutant genes involved,
ade1 and ade2, have the same phenotype (red, adenine-dependent).
Note how different the patterns of phenotypes is form the two
different phenotype case, in spite of the same underlying
segregation pattern. You can also use this as an example of the
red phenotype at one locus being epistatic to the cream phenotype
at the other locus.

When red, adenine-requiring mutants were first discovered and
studied, it was this test that demonstrated that there are two
genes involved. This allelism test is the basis for assigning
and newly discovered mutant to an already known gene.

In this experiment the students start with F1 haploids
(gametes) to obtain the F2 generation, with no statistical
sampling complications by setting up the following grid of
crosses: